The human TCL-1 gene maps at chromosome 14q32.1 and is activated in T cell leukemias and lymphomas by either chromosome translocations or inversions that juxtapose the TCL-1 gene to the alpha/delta or beta locus of the T cell receptor. The hTCL-1 protein is 41% identical and 61% similar to the hMTCP-1 protein on chromosome Xq28 which is also involved in T cell lymphoproliferative diseases. The hTCL-1 and hMTCP-1 proteins may be new members of an oncogene family involved in lymphoid proliferation and/or survival and in T cell malignancies. The major role of the Protein Chemistry Core Laboratory in this project is to produce and purify the TCL-1 and MTCP-1 proteins in milligram amounts which are necessary to conduct functional analysis and three- dimensional structural analysis of these proteins. The hTCL-1 and hMTCP-1 proteins have been expressed in C. coli both with and without 6xHis tag sequence, and the recombinant proteins have been purified to homogeneity in milligram amounts. The hTCL-1 and hMTCP-1 proteins will be produced using a eukaryotic expression system, purified to homogeneity in milligram amounts, and their properties compared to the E. coli produced proteins. The E. coli system will also be used for the preparation of mutant forms of hTCL-1. Core B will conduct studies to characterize the properties of all recombinant TCL-1 and MTCP-1 proteins produced in this laboratory using the techniques of N-terminal sequence analysis, electrospray mass spectrometry (ES-MS), isoelectric focusing (IEF), gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). The Protein Chemistry Core will map putative nucleotide binding sites and the exposed hydrophobic sites of both TCL-1 and MTCP-1 proteins using photoincorporation of [32P]-labeled and fluorescent probes, fragmentation by chemical or enzymatic methods, separation by HPLC, and peptide sequencing. Core B will conduct studies to determine the binding of wild-type and mutant forms of TCL-1 and MTCP-1 to interacting proteins such as EWS and TAF/II68 and also further studies to identify new molecular partners of TCL-1 and MTCP-1 using the biosensor technology. The Core will also prepare monoclonal and polyclonal antibodies to TCL-1 and MTCP-1, mutant forms, peptides, and interacting proteins. Core B will prepare by solid phase synthesis all peptide analogs of TCL-1 and MTCP-1 designed by molecular modeling to act a potential inhibitors of their protein function.